Colorectal cancer is the 2nd leading cause of cancer-related mortality, producing ~10% of cancer-related deaths. Mortality universally reflects metastatic disease, which develops in 50% of patients, and remains incurable by current modalities. There is a developing focus on exploiting the efficacy and specificity of the immune system to prevent and treat cancer. In that context, cancer vaccines that mobilize immune responses to tumor antigens in patients fail to eradicate established metastases, reflecting immunosuppressive networks that produce tolerance against antigen-specific responses. Adoptive T cell therapy, in which tumor-specific T cells are expanded ex vivo and re-introduced into patients, has emerged as an approach to eradicate metastases, producing regression of tumors in patients with melanoma, leukemia, and neuroblastoma. However, this approach has been unsuccessful in colorectal cancer due to toxicities in normal tissues reflecting inadequate compartmentalization of target antigens. This proposal focuses on eradicating colorectal cancer metastases employing genetically engineered T cells targeted to the tumor antigen guanylyl cyclase C (GUCY2C) by chimeric antigen receptors. GUCY2C is the index example of a class of proteins termed cancer mucosa antigens, expressed selectively in mucosal compartments and by derivative tumors, with unique characteristics which make them effective as targets in cancer immunotherapy. GUCY2C is selectively expressed in apical membranes of intestinal epithelial cells in the mucosal compartment, which is structurally, functionally, and immunologically sequestered from the systemic compartment by the intestinal barrier. Moreover, GUCY2C is universally over-expressed by colorectal cancer metastases. GUCY2C-based vaccines prevent colorectal cancer metastases in mice and have advanced into phase I clinical trials. Here, mouse T cells will be engineered to express GUCY2C-specific chimeric antigen receptors, which fuse intracellular T cell signaling domains to antigen-recognizing single-chain fragment variable regions from GUCY2C-specific monoclonal antibodies. These receptors offer a substantial advantage compared to endogenous antigen- specific T cells because they recognize native antigens independent of MHC, with universal applicability across all patients. Importantly, MHC-independent recognition of native antigen provides a unique opportunity to target T cells to metastases in the systemic compartment without inducing intestinal toxicity, reflecting sequestration of GUCY2C in the mucosa. Here, study outcomes will determine the ability of T cells expressing GUCY2C- specifc chimeric receptors to undergo proliferation, secrete cytokines, produce pro-survival signaling, and induce tumor cell cytotoxicity induced by antigen in vitro. Moreover, the ability of these engineered T cells to promote durable in vivo regression of parenchymal metastases in the absence of toxicities in normal tissues will be defined. Successful completion of these studies will position GUCY2C-targeted T cells for translation to treat established metastatic disease in patients with advanced colorectal cancer.